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Quantitation of the mRNA expression of the epidermal growth factor system: Selective induction of heparin-binding epidermal growth factor–like growth factor and amphiregulin expression by growth factor stimulation of prostate stromal cells
Quantitation of the mRNA expression of the epidermal growth factor system: Selective induction of heparinbinding epidermal growth factor–like growth factor and amphiregulin expression by growth factor stimulation of prostate stromal cells BOE SANDAHL SØRENSEN, NIELS TØRRING, MUSTAFA VAKUR BOR, and EBBA NEXO AARHUS, DENMARK
The epidermal growth factor (EGF) system is a rapidly expanding system of growth factors involved in many aspects of normal and cancerous growth. We have developed a method for the quantitation of mRNA coding for all six growth factors activating the human EGF receptor (HER-1) and for the quantitation of mRNA for the receptors HER-1 and its preferred dimerization partner, HER-2. The method is based on the generation of specific RNA standards, which are amplified by reverse transcriptase–polymerase chain reaction (RT-PCR) with the sample RNA and a set of calibrators. The resulting calibration curve is used to quantitate the unknown samples, which require only a single RT-PCR reaction. Our method has the advantage that quantitation is based on coamplification of an internal RNA standard, thereby controlling both the PCR and RT reactions. In addition, the RNA standards for all growth factors and receptors are combined in a single RT reaction, which minimizes variation and allows the quantitation of all eight mRNA species with only 0.1 µg RNA. This makes the method suitable for studies in which the supply of material is limited. The developed method has enabled us to demonstrate that prostate stromal cells in primary culture express EGF, heparin-binding EGF (HB-EGF), amphiregulin, betacellulin, and epiregulin as well as the HER-1 and HER-2 receptors, whereas no transforming growth factor-α mRNA is found. Furthermore, activation of the EGF system in these cells by stimulation with HB-EGF or EGF in mitogenic doses causes a selective increase in the expression of amphiregulin and HB-EGF mRNA (more than 15-fold and 25-fold, respectively), whereas there is no increase in the expression of mRNA for the other growth factors or receptors. In accord with the increase in amphiregulin mRNA, the amount of amphiregulin peptide released from the cells is also increased. The selective induction of amphiregulin and HB-EGF by growth factor stimulation may represent a mechanism to amplify the initial growth factor signal in prostate stromal cells. (J Lab Clin Med 2000;136:209-17) Abbreviations: dATP = deoxyadenosine triphosphate; dCTP = deoxycytidine triphosphate; dGTP = deoxyguanosine triphosphate; dTTP = deoxythymidine triphosphate; EGF = epidermal growth factor; EGTA = ethyleneglycol-bis (β-aminoethylether)-N,N,N´,N´-tetraacetic acid; ELISA = enzyme-linked immunosorbent assay; HB-EGF = heparin-binding epidermal growth factor-like growth factor; HEPES = N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid; HER = human epidermal growth factor receptor; PBS = phosphate-buffered saline solution; PIPES = piperazine-N,N ’- bis (2-ethanesulfonic acid); RT-PCR = reverse transcriptase–polymerase chain reaction; TGF-α = transforming growth factor-α From the Department of Clinical Biochemistry, AKH, Aarhus University Hospital. Supported by the Danish Medical Research Council, the Danish Cancer Society, the Danish Cancer Research Foundation, and the Clinical Research Unit of the Danish Cancer Society, Aarhus, and as part of the Aarhus University Novo Nordic center for research in growth and regeneration. Submitted for publication January 14, 2000; revision submitted April 24, 2000; accepted May 11, 2000.
Reprint requests: Boe Sandahl Sørensen, Department of Clinical Biochemistry, AKH, Aarhus University Hospital, Nørrebrogade 44, DK8000 Aarhus C, Denmark. Copyright © 2000 by Mosby, Inc. 0022-2143/2000 $12.00 + 0 5/1/108753 doi:10.1067/mlc.2000.108753
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Originally the EGF system was considered to consist of one receptor (HER-1) and one ligand, EGF. During the last few years five additional growth factors activating HER-1 have been described, together with additional receptors. Among these receptors, HER-2 is the preferred dimerization partner for HER-1.1 The five additional ligands are betacellulin, HB-EGF, epiregulin, TGF-α, and amphiregulin.2 It is well known that activation of the EGF system is of importance both for growth and for differentiation of cells. Lack of activity in the system is incompatible with life, and increased activity in the system is a common feature of malignant growth.3 Despite this general knowledge on the importance of the system, the relative impact of the many ligands of the EGF system is not known. Investigation of this area is not an easy task. The system is considered to act locally as paracrine and autocrine growth factors; for this reason there is a need for reliable analyses that can determine the expression of the EGF system in minute amounts of tissue or in cultured cells. One of the organs believed to be influenced by the EGF system is the prostate gland.4 Growth of the prostate can lead either to a hyperplastic growth of the gland or to prostate cancer. Growth factors from the EGF system have been shown to be responsible for autocrine stimulation of prostate cancer cells.5 Furthermore, stimulation with EGF induces proliferation of epithelial cells derived from the prostate.6 Proliferation of stromal cells from the prostate has also been shown to be increased by growth factor stimulation.7 In the prostate the EGF system has also been suggested to play an important role for stroma-epithelium interactions.8 To further explore the role of the EGF system, our aim was to develop a method that makes it possible to quantitate the expression of all of the HER-1–activating ligands, of HER-1, and of its preferred heterodimerization partner, HER-2, in prostate stromal cells grown in primary culture. In addition, we wanted to investigate the regulation of the expression of the EGF system during growth factor–induced proliferation. Competitive RT-PCR with an RNA standard that is amplified together with the sample RNA is a sensitive method for mRNA quantitation. Previously we have shown that the method can be refined by inclusion of a set of calibrator RNA samples and development of a calibration curve.9 In the present article we extend this method to include a quantitation of all of the HER-1 activating growth factors and two receptors from the EGF system in human beings. Employing this method, we demonstrate that prostatic stromal cells express all of the ligands examined except TGF-α and all of the receptors investigated (HER-1 and HER-2). We demonstrate a selective induc-
tion of amphiregulin and HB-EGF on stimulation with EGF or HB-EGF. METHODS Cell isolation and culture conditions. Primary cultures of prostate stromal cells were established from human prostate tissue isolated by transurethral resection. Characterization and culturing of the prostate stromal cells was performed as we have previously described,10 according to the method of Zhang et al.11 The cells were cultured in the following serum-containing medium: MCDB 131 (Sigma), 15% heat-inactivated and calcium-stripped horse serum, 1% penicillin/streptomycin, 1% nonessential amino acids, 10 mmol/L HEPES (all from Biological Industries), 10 µg/mL transferrin, 5 µg/mL human insulin (both from Boehringer Mannheim), 5 ng/mL sodium selenite, 0.1 µmol/L estradiol, 0.1 µmol/L dexamethasone (all from Sigma), 1 ng/mL basic fibroblast growth factor, and 0.1 ng/mL EGF (both from Biomol). Cells growing out from tissue pieces were detached by trypsination and passed. In brief, for determination of cell proliferation, 2000 cells/cm2 were seeded in 96-well microtiter trays and grown for 1 day in the serum-containing medium described above. The cells were then starved for 2 days in serum-free medium consisting of MCDB 131 medium (Sigma), 0.1% wt/vol human albumin (Behringwerke), 1% penicillin/streptomycin, 10 mmol/L HEPES, 10 µg/mL transferrin, 2,5 µg/mL human insulin, and 5 ng/mL sodium selenite. Finally, the cells were stimulated with 1 nmol/L HBEGF or 1 nmol/L EGF for 24 hours. Proliferation was measured with the MTT assay (Promega) according to the manufacturer’s instructions. The HCV29 cell line used as calibrator in the mRNA quantitation method was from the Fibiger Institute, Denmark. This cell line is of urothelial origin, characterized by its immortalized growth pattern, and histologically graded as tumor grade two.12 RNA isolation. Cells in third passage were seeded in tissue culture dishes (Greinar) at a density of 2000 cells/cm2 and cultured in the serum-containing medium described above. After 72 hours the medium was changed to the previously described serum-free medium, and the cells were kept in this medium for 48 hours. Cells were stimulated with 1 nmol/L HB-EGF (R&D Systems) or 1 nmol/L EGF (Oncogene) in this medium. The cells were detached from the culture flasks by scraping and were collected by centrifugation at 1500 rpm for 5 minutes. Total RNA was isolated with the Purescript kit (Gentra) by following the instructions provided by the manufacturer. After purification, the concentration and purity of the RNA preparations were analyzed by optical density at wavelengths of 260 and 280 nm (GeneQuant II; Pharmacia Biotech), as previously described.13 Generation of internal RNA standards. RNA internal standards were prepared by modification of previously described methods.9,14 A segment of a 0.6 kb EcoRI-BamHI fragment (spacer DNA) of the v-erb B oncogene (Clontech) was PCR amplified with two composite primers (illustrated in Fig 1). One primer contained at the 5´ end the T7 RNA polymerase promotor sequence followed by one of the genespecific primer sequences and at the 3´ end a region hybridizing to the spacer DNA sequence. The other primer was com-
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posed of a stretch of 20 T nucleotides at the 5´ end followed by the other gene-specific primer and at the 3´ end a sequence that hybridized to the spacer DNA. A PCR was performed that contained 1 ng of the spacer DNA, 10 pmol of each of the two composite primers, 1.5 mmol/L MgCl2, 0.2 mmol/L deoxyribonucleoside triphophates, and 2.5 U Taq DNA polymerase (Pharmacia). A Perkin Elmer thermal cycler was used with an amplification profile of 1 minute at 94°C, 30 seconds at 65°C, and 90 seconds at 72°C (an initial heating to 94°C for 3 minutes was performed). Thirty cycles were conducted, and at the end the samples were extended for 7 minutes at 72°C. A single DNA band of the correct size was verified by gel electrophoresis (data not shown). RNA internal standard was obtained by incubation of 1 µg of the PCR product with 40 U of T7 RNA polymerase (Boehringer Mannheim) in the buffer supplied by the manufacturer for 2 hours at 37°C. DNA was digested with ribonuclease-free DNase I (Promega) followed by extraction with water-saturated phenol/chloroform (24:1). RNA in the water phase was precipitated by the addition of 1/10 vol of 4 mol/L NaCl and 2.5 vol of 96% ethanol. The RNA was recovered by centrifugation for 15 minutes at 14,000 rpm in an Eppendorf centrifuge and washed with 1 mL 70% ethanol. The RNA pellet was redissolved in nuclease-free water and passed through a spin column (Chromaspin, Clontech) to remove free nucleotides, and finally the RNA was quantitated by spectrophotometry.13 PCR products were not obtained in the absence of reverse transcription, demonstrating the absence of contaminating DNA in the RNA standards (data not shown). By designing the composite primers to hybridize to different positions on the spacer DNA, it was possible to produce RNA internal standards with suitable sizes. The sizes of the internal RNA standards were adjusted according to the sizes of the mRNA targets, resulting in size differences between 50 and 200 bp for the eight mRNA species investigated. Internal RNA standards were generated for EGF, TGF-α, amphiregulin, HB-EGF, betacellulin, epiregulin, HER-1, and HER-2, and the sizes of the RT-PCR products originating from the internal RNA standards and the corresponding mRNAs are presented in Table I together with the sequences of the gene-specific primers used. Quantitative RT-PCR. Calibrators for the quantitative RTPCR assays were obtained from a pool of total RNA isolated from the urothelial cell line HCV29. This RNA was diluted in water to give calibrators with the following RNA concentrations: 1 µg/µL, 750 ng/µL, 500 ng/µL, 250 ng/µL, 100 ng/µL, 75 ng/µL, 50 ng/µL, 25 ng/µL, 10 ng/µL, 8 ng/µL. The HCV29 cell line was chosen as calibrator because it expresses all of the mRNA species investigated at a sufficiently high concentration for quantitation. The absolute concentrations of the specific mRNAs in the HCV29 calibrator RNA were determined by competitive RT-PCR. In brief, a fixed amount of RNA standard was coamplified with a dilution series of the calibrator RNA (RT-PCR conditions were as described below). The intensities of the bands originating from the RNA standard and the specific mRNA in the calibrator were plotted against the amounts of calibrator used. By this method an amount of calibrator was identified in which equal amounts
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Fig 1. Schematic illustration of the generation of RNA standards. One primer consists of the T7 RNA polymerase promotor sequence (T7), a gene-specific primer sequence (GS1), and a region hybridizing to the spacer DNA (HYB). The other primer is composed of a region of 20 T nucleotides (T20), the other gene-specific primer sequence (GS2), and a region hybridizing to the spacer DNA (HYB). A DNA molecule is generated by PCR, and in vitro transcription of this DNA gives rise to the RNA standard, which has incorporated the gene-specific primer sequence (GS1), the complementary sequence of the other gene-specific primer (GS2), and a tail of 20 A nucleotides (A20). GS1 and GS2 represent the gene-specific primers, and Table I gives the sequences of all eight pairs of gene-specific primers used.
of RT-PCR products from the specific mRNA and the RNA standard were generated. In this amount of calibrator the content of the specific mRNA equals the amount of RNA standard added. Because the RNA standard was present in a known amount, this made it possible to find the absolute mRNA concentrations in the calibrator. Gel electrophoresis, scanning of bands, and correction for differences in the sizes of target and standard bands were as described below. In the quantitative assays described in the present article, a fixed amount of each of the eight internal RNA standards was combined with the sample RNA to be analyzed and to a set of the HCV29 RNA calibrators, followed by RT-PCR amplification. RNA (0.1 µg) was mixed with the eight different RNA standards in a mixture of 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 6.25 mmol/L MgCl2, 1 U/µL RNase inhibitor, 1 mmol/L deoxyribonucleoside triphosphates (dATP, dCTP, dTTP, dGTP), 2.5 µmol/L of a 16 mer d(T)16 oligonucleotide primer, and 2.5 U/µL reverse transcriptase in a reaction mixture of 20 µL (all reagents from Perkin-Elmer). Reverse transcription was performed by incubation at 42°C for 30 minutes. A 2.5 µL sample of the reaction mixture was used for PCR in a total volume of 25 µL containing 50 mmol/L KCl, 10 mmol/L Tris-HCl (pH 9.0), 1.5 mmol/L MgCl2, 0.2 mmol/L deoxyribonucleoside triphosphates (dATP, dCTP, dTTP, dGTP), 1.25 U of Taq polymerase (Pharmacia), and the amount of primer shown in Table I. PCR was performed in a Perkin Elmer thermal cycler with the following standard cycling parameters: 94°C for 1 minute, annealing temperature (presented in Table I) for 30 seconds, and 72°C for 90 seconds (initially the samples were dena-
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Table I. Primer sequences and sizes of RT-PCR products Gene-specific primers
Target size
Standard size
Amphiregulin
5´-TTGATACTCGGCTCAGGCC-3´ 5´-CTACTGTCAATCATGCTGTGA-3´ (4 pmol/60°C/39 cycles)
539 bp
345 bp
HB-EGF
5´-GATGGTTGTGTGGTCATAGGT-3´ 5´-TGGCTGCAGTTCTCTCGGC-3´ (10 pmol/62°C/35 cycles)
449 bp
340 bp
Betacellulin
5´-CTGCAAAGTGCCTTGCTCA-3´ 5´-TGACTAGTAATCCTGGTGAC-3´ (10 pmol/59°C/33 cycles)
251 bp
315 bp
Epiregulin
5´-AATGTAACTCCACTGTTCTCC-3´ 5´-CCTTCTCCCATATGAACAAGA-3´ (10 pmol/59°C/33 cycles)
357 bp
418 bp
EGF
5´-AGCAATTGGTGGTGGATG-3´ 5´-ACTCTTTGCAAAAGTTGTC-3´ (5 pmol/54°C/35 cycles)
103 bp
163 bp
TGF-α
5´-GCCCGCCCGTAAAATGGTCCCCTC-3´ 5´-GTCCACCTGGCCAAACTCCTCCTCTGGG-3´ (2.5 pmol/70°C/32 cycles)
528 bp
357 bp
HER-1
5´-GAGAGGAGAACTGCCAGAA-3´ 5´-GTAGCATTTATGGAGAGTG-3´ (25 pmol/57°C/30 cycles)
454 bp
314 bp
HER-2
5´-AGATGTTCGGCCCCAGCCCCCTT-3´ 5´-GTGGAGCCCCCCGCTCTGGTG-3´ (10 pmol/68°C/30 cycles)
272 bp
420 bp
Items in parentheses contain the following information: primer concentration/annealing temperature/PCR cycles.
tured at 94°C for 3 minutes). After the number of cycles used for each of the PCR products (Table I), the PCR products were extended for 7 minutes at 72°C. PCR products were analyzed by electrophoresis in a 2% agarose gel (Meda). The intensities of the bands were determined by computer scanning with the Gel Doc 1000 system (BioRad) and the molecular analyst software (BioRad). Intensities of the target and standard bands were corrected for differences in size of the RT-PCR products, because binding of ethidium bromide is dependent on DNA size. The ratio of the amount of PCR product originating from the target mRNA and the internal standard RNA was determined for each of the calibrators. This ratio was plotted against the amount of the specific mRNA present in each calibrator, thereby generating a calibration curve. By use of the calibration curve, the ratio of the PCR products from target and internal standard RNA obtained from the unknown samples could be used to quantitate the specific mRNAs. Samples exceeding the standard curve were diluted in water before repeated analysis. Coefficients of variation of the calibrated quantitative assays ranged between 9% and 21%, as is typical for this type of assay. Equal efficiency of the RT-PCR amplification of target mRNA and corresponding internal RNA standard was demonstrated by RT-PCR amplification of mixtures of the two molecules with decreasing numbers of cycles (data not shown). The identity of all PCR products was verified by sequencing (ABI Prism 310; Perkin Elmer).
Peptide expression. Cells in fourth or fifth passage were seeded in tissue culture flasks (T175; NUNC) at a density of 2000 cells per cm2 and cultured in the serum-containing medium described above. After 48 hours the medium was changed to serum-free medium. Forty-eight hours later, 1 nmol/L HB-EGF or 1 nmol/L EGF was added to the medium and the cells were cultured for an additional 96 hours. The medium was removed and filtered through a 0.22µm filter (Gelman Sciences), concentrated 10-fold by lyophilization, and dialyzed (cut off value of 3.5 kd) for 48 hours in PBS containing 0.1% bovine albumin (Sigma). Cells were washed in ice-cold PBS and scraped off. The cells were isolated by centrifugation at 1500 rpm for 8 minutes at 4°C before being stored at –20°C. After thawing, 0.3 mL of homogenization buffer (pH 7.4) containing 10 mmol/L PIPES (Sigma), 1 mmol/L EGTA (Sigma), 3 mmol/L MgCl2, 400 mmol/L NaCl, and complete protease inhibitor (Boehringer Mannheim) was added to the cells, and the suspension was sonicated three times for 10 seconds before centrifugation at 20,000g for 40 minutes at 4°C. The supernatant was isolated (supernatant I), and the pellet was suspended in 0.3 mL homogenization buffer containing 2% Triton X-100 and sonicated. The suspension was left overnight at 4°C and centrifugated as indicated above. The supernatant was isolated (supernatant II) and stored at –20°C before analysis. The concentration of growth factors in the cell supernatant
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A Fig 2. RT-PCR coamplification of mRNA and internal RNA standards from the EGF system. Calibrator RNA (RNA from the HCV29 cell line) was RT-PCR amplified with internal RNA standard. Lanes 1 through 8 represent HER-1, HER-2, amphiregulin, betacellulin, EGF, epiregulin, HB-EGF, and TGF-α, respectively (DNA sizes are presented in Table I). M is the size marker (φX174 DNA digested with Hae III).
and conditioned medium was determined by traditional sandwich ELISA technique. Amphiregulin was measured with the MAB-262 (capture antibody) and the AB-262-NA (detecting antibody), both from R&D Systems. Recombinant human amphiregulin 262-AR-100 (R&D Systems) was used as calibrator. EGF was measured with the rabbit anti-mouse IgG1 (Z0013-DAKO), the anti human EGF Ab-1 (GF01, Calbiochem) as capture antibody, and a polyclonal rabbit antihuman EGF antibody as detecting antibody.15 Recombinant human EGF (Oncogene) was used as calibrator. Protein concentrations of the supernatants were determined by the BCA protein assay (Pierce).
B
RESULTS Quantitation of mRNA expression from the EGF system.
Quantitative RT-PCR based methods were developed for EGF, amphiregulin, TGF-α, HB-EGF, betacellulin, epiregulin, HER-1, and HER-2. Primer sequences, annealing temperatures, numbers of PCR cycles, and sizes of the RT-PCR products obtained from the genespecific mRNAs and internal RNA standards for the members of the EGF system are presented in Table I. Fig 2 shows all eight target and corresponding internal RNA standard pairs investigated. Fig 3, A, shows the RT-PCR amplification of HBEGF mRNA and the corresponding internal RNA standard as a representative example of the quantitative mRNA assay. In brief, in the quantitative assay, a mixture of all eight internal RNA standards was added to both the RNA samples (0.1 µg) and the calibrators, and a single reverse transcription reaction was performed. This cDNA was used as the template for all eight specific PCR reactions. A calibration curve was generated by plotting the ratio of the intensities of the RT-PCR bands from HB-EGF mRNA and internal RNA standard against the amount of the specific mRNA in the individual calibrators. Fig 3, B, and Fig 3, C, show the gel image and calibration curve, respectively, of the quantitative HB-EGF mRNA assay.
C Fig 3. Method for quantitation of mRNA from the EGF system. A, Schematic illustration of the PCR amplification of HB-EGF mRNA and standard RNA with the same primer pair (P1 and P2) shown as an example. B, Gel showing the calibration curve obtained by RTPCR amplification of serial dilutions of calibrator RNA combined with a fixed amount of internal RNA standard. Lanes 1 through 10 represent 1.0 µg, 0.75 µg, 0.5 µg, 0.25 µg, 0.1 µg, 0.075 µg, 0.05 µg, 0.025 µg, 0.01 µg, and 0.008 µg, respectively. Lane 11 is a control with standard RNA but without calibrator RNA. C, Calibration curve based on scanning of the gel in B. The ratios of the RT-PCR products from HB-EGF mRNA and internal RNA standard are depicted as a function of the amount of HB-EGF mRNA present in the added calibrator RNA samples. The standard curve is obtained by linear regression (y = 1.0x + 19.0).
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A
B Fig 4. Selective induction of HB-EGF and amphiregulin expression by growth factor stimulation of prostate stromal cells. A, Prostate stromal cells were kept in minimal essential medium for 48 hours, and the mRNA expression of growth factors and receptors from the EGF system was determined. Each bar represents the mean expression level of four individual experiments (standard deviation is indicated). B, The cells were incubated in the presence or absence of 1 nmol/L HB-EGF for 48 hours, and the expression of growth factors and receptors was determined. The induction of mRNA expression is presented as the ratio between the mRNA expression in the HB-EGF stimulated and unstimulated cultures (ratio of the mean expression in two stimulated and two unstimulated cultures). The experiments presented in A and B are representative examples of three independent experiments. The inserted gel picture shows representative RT-PCR amplification data illustrating the coamplification of a fixed amount of HB-EGF internal RNA standard and HB-EGF mRNA from RNA isolated from HB-EGF treated and control cells (lanes 1 and , respectively). For the actual quantitation, calibrators would be included and a standard curve would be generated (as presented in Fig 3).
HB-EGF and amphiregulin mRNA is induced by stimulation with HB-EGF or EGF. Prostate stromal cells grown
in primary cultures and kept in a serum-depleted medium for 48 hours express mRNA for all the growth factors and receptors examined except TGF-α. The expression was lowest for amphiregulin, while comparable expression was observed for betacellulin, epiregulin, EGF. and HB-EGF (Fig 4, A). The level of mRNA expression for the two receptors was slightly higher than the expression for the growth factors (Fig 4, A).
Prostate stromal cells were grown in the absence or presence of HB-EGF or EGF (both 1 nmol/L) for 48 hours. In Fig 4, B, the levels of induction of growth factor and receptor mRNA expression after stimulation with HB-EGF are presented as mRNA expression in the stimulated cells divided by expression in the unstimulated cells. This demonstrates that a selective induction of amphiregulin and HB-EGF mRNA takes place. A similar result was observed after 24 hours of incubation. None of the other ligands and none of the
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Fig 5. Induction of amphiregulin peptide by HB-EGF stimulation of prostate stromal cells. Prostate stromal cells were kept in minimal essential medium for 96 hours in the presence or absence of 1 nmol/L HB-EGF. Cells and medium from the cells were isolated, and the amount of amphiregulin and EGF peptide was determined as described in Methods. During the experiment there was a 40 % increase in cell number of the HB-EGF–treated cells. The peptide concentrations of the HB-EGF–treated cells were adjusted to account for this increase in cell number. No EGF was found in the medium of either control or HB-EGF–stimulated cells. Shown is a representative example. The experiment was repeated twice with similar results.
Fig 6. EGF- and HB-EGF–stimulated proliferation of prostate stromal cells. Cells were serum-starved for 48 hours and then grown for 96 hours with either 1 nmol/L HB-EGF or 1 nmol/L EGF or in the absence of growth factor (control cells). Proliferation was determined by the MTT assay. Data are presented as fold increase in proliferation as compared with the proliferation of the control cells. Each column represents the mean value of 16 colonies (standard deviation indicated). There was a significant increase in proliferation after EGF and HB-EGF treatment (P < .01) as compared with the control cells. The experiment was repeated three times, and a representative example is shown.
receptors examined increased after stimulation with HB-EGF (Fig 4, B). Similar results were obtained by stimulation with EGF (data not shown).
stimulated the growth of prostate stromal cells in primary cultures (P < .01). Fig 6 shows the results obtained after growth factor stimulation for 96 hours. The figure presents the mean ± SD values for 16 individual cultures. Similar results were obtained in two additional experiments that used different primary cultures.
Induction of amphiregulin peptide by HB-EGF stimulation. The concentrations of amphiregulin and EGF pro-
tein were determined in both culture medium and cells after 96 hours in serum-free medium with or without 1 nmol/L HB-EGF. The control cells contained comparable amounts of EGF and amphiregulin (Fig 5). No EGF was detected in the medium, whereas a total of 140 fmol amphiregulin was measured in the 40 mL of medium collected from the control cells. The amount of EGF did not increase on stimulation with HB-EGF. Likewise, the concentration of amphiregulin in the cells was not affected by HB-EGF. In contrast, the concentration and the total amount of amphiregulin in the medium increased to 240% after HB-EGF treatment (340 fmol in the 40 mL medium). Correcting for the increase in cell number (40%), we found the amount of amphiregulin released per cell to increase to 170% of the control level after treatment of the cells with HB-EGF (Fig 5). All protein and mRNA quantitations presented were performed on primary cultures from the same donor. Similar results were found in experiments performed on primary cultures obtained from two different donors. HB-EGF and EGF stimulate proliferation of prostate stromal cells. The mitogenic potential of the HB-EGF and
EGF doses used to induce mRNA expression of HBEGF and amphiregulin was investigated. Equimolar amounts (1 nmol/L) of HB-EGF or EGF significantly
DISCUSSION
Because of the complexity of the EGF system, refined methods are required so that the mRNA expression of several members of the family can be investigated simultaneously. We report a new method for quantitating the mRNA expression of the six HER-1 activating growth factors as well as HER-1 and its preferred heterodimerization partner, HER-2. This calibrated RT-PCR reaction has the advantage that only a single PCR reaction is required to analyze each mRNA. Imprecision is minimized by our procedure, in which all eight RNA standards are combined with the sample RNA before reverse transcription, thereby generating a common cDNA sample that can be used for all eight specific PCR reactions. A further advantage is that only a 0.1-µg sample of RNA is required for quantitation of all eight mRNA species. This makes the method suitable for clinical applications in which limited material is available. The design of the assay makes it possible to include control samples, which can be used to monitor the performance of the assay over time. An important prerequisite for correct quantitation by the assays
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described here is that the RNA standard and the target mRNA are RT-PCR anplified with the same efficiency. This can be verified by demonstrating that the ratio between the target and standard RT-PCR products from RNA samples remains unaffected when the cycle number in the PCR amplification is decreased.9 Growth of the prostate as observed in both benign prostatic hyperplasia and prostate cancer represents important clinical problems. However, only limited information is available regarding the expression of the components of the EGF system in human prostate stromal cells and the regulation of their expression during growth factor–induced proliferation. Our new method has enabled us to demonstrate for the first time that five of the six EGF receptor ligands, as well as the two receptors examined, are expressed in primary cultures of prostate stromal cells. We further demonstrate that stimulation of the cells with mitogenic concentrations of either HB-EGF or EGF selectively increases the expression of HB-EGF and amphiregulin by more than 25-fold and 15-fold, respectively. Growth factor stimulation had no effect on mRNA expression of the HER1 and HER-2 receptors, which indicates that induction of the EGF system in prostate stromal cells is mediated at the growth factor level rather than by induction of the HER-1 and HER-2 receptors. In accord with the amphiregulin mRNA induction on stimulation with growth factor, the amphiregulin protein level was also increased in the medium from the cell cultures, as determined by ELISA. A suitable assay for quantitation of the other induced growth factor (HBEGF) at the protein level was not available. Both HBEGF and amphiregulin are distinguished from the other ligands from the EGF system by their heparin-binding ability, suggesting that this subgroup of growth factors might play a special role in the induction of growth factors after growth factor stimulation of prostate stromal cells. At present the biologic significance of this induction is not known, but we have demonstrated that exogenous HB-EGF (the present study) and amphiregulin10 induce proliferation of prostate stromal cells. Autoinduction of growth factors from the EGF system has been shown to occur in a few other cell types such as keratinocytes, intestinal cells, and fetal vascular smooth muscle cells.16-18 In one study, four of the six ligands were investigated, and it was reported that in growth factor–treated keratinocytes, all of the growth factors examined were induced.16 HB-EGF and amphiregulin were induced to the highest level, whereas betacellulin and TGF-α were induced to a lower degree. In contrast, we found that in primary cultures of human prostate stromal cells, the mRNA induction is restricted to HB-EGF and amphiregulin. These two studies demonstrate that in different cell types, differ-
ences exist with respect to the induction of ligands from the EGF system. A consequence of the growth factor induction reported here might be that the signal from the initial growth factor stimulation is amplified and sustained, thereby promoting autocrine growth of the stromal cells. In addition, our observation that amphiregulin is secreted to the culture medium of the prostate stromal cells suggests that the increased growth factor expression is also responsible for mediating paracrine stimulation of the prostatic epithelium. This possibility is in accord with previous studies showing a high expression of HER1 in the basal epithelial cell layer of the prostate,19 which is in close approximation to the stromal cells. This concept is further supported by recent findings showing that amphiregulin and HB-EGF are coordinately regulated in prostate smooth muscle cells and are likely to be involved in signaling between the smooth muscle cells and the epithelial cells of the prostate.20 In conclusion, we present a method whereby the mRNA expression from all six HER-1 ligands, as well as the HER-1 and HER-2 receptors, can be quantitated simultaneously in a limited amount of RNA (0.1 µg). The method can easily be applied to other studies in which the simultaneous quantitation of several mRNA species is important. The developed method has enabled us to demonstrate that growth factor activation of prostate stromal cells grown in primary culture results in a selective induction of amphiregulin and HB-EGF mRNA. We thank Klaus Møller-Ernst Jensen for providing the human prostate tissue. We also thank Birgit Westh Mortensen, Alice Villemoes, and Inger Marie Jensen for technical assistance.
REFERENCES
1. Graus Porta D, Beerli RR, Daly JM, Hynes NE. ErbB-2, the preferred heterodimerization partner of all ErbB receptors, is a mediator of lateral signaling. EMBO J 1997;16:1647-55. 2. Alroy I, Yarden Y. The ErbB signaling network in embryogenesis and oncogenesis: signal diversification through combinatorial ligand-receptor interactions. FEBS Lett 1997;410:83-6. 3. Salomon DS, Brandt R, Ciardiello F, Normanno N. Epidermal growth factor-related peptides and their receptors in human malignancies. Crit Rev Oncol Hematol 1995;19:183232. 4. Culig Z, Hobisch A, Cronauer MV, Radmayr C, Hittmair A, Zhang J, et al. Regulation of prostatic growth and function by peptide growth factors. Prostate 1996;28:392-405. 5. Connolly JM, Rose DP. Regulation of DU145 human prostate cancer cell proliferation by insulin-like growth factors and its interaction with the epidermal growth factor autocrine loop. Prostate 1994;24:167-75. 6. McKeehan WL, Adams PS, Rosser MP. Direct mitogenic effects of insulin, epidermal growth factor, glucocorticoid, cholera toxin, unknown pituitary factors and possibly pro-
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lactin, but not androgen, on normal rat prostate epithelial cells in serum-free, primary cell culture. Cancer Res 1984; 44:1998-2010. Levine AC, Ren M, Huber GK, Kirschenbaum A. The effect of androgen, estrogen, and growth factors on the proliferation of cultured fibroblasts derived from human fetal and adult prostates. Endocrinology 1992;130:2413-9. Freeman MR, Paul S, Kaefer M, Ishikawa M, Adam RM, Renshaw AA, et al. Heparin-binding EGF-like growth factor in the human prostate: synthesis predominantly by interstitial and vascular smooth muscle cells and action as a carcinoma cell mitogen. J Cell Biochem 1998;68:328-38. Bor MV, Sorensen BS, Rammer P, Nexo E. Calibrated userfriendly reverse transcriptase-PCR assay: quantitation of epidermal growth factor receptor mRNA. Clin Chem 1998;44: 1154-60. Torring N, Sorensen BS, Bösch ST, Klocker H, Nexo E. Amphiregulin is expressed in primary cultures of prostate myofibroblasts, fibroblasts, epithelial cells, and in prostate tissue. Prostate Cancer and Prostatic Diseases 1998;5:262-7. Zhang J, Hess MW, Thurnher M, Hobisch A, Radmayr C, Cronauer MV, et al. Human prostatic smooth muscle cells in culture: estradiol enhances expression of smooth muscle cellspecific markers. Prostate 1997;30:117-29. Christensen B, Kieler J, Vilien M, Don P, Wang CY, Wolf H. A classification of human urothelial cells propagated in vitro. Anticancer Res 1984;4:319-37. Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. New York: Cold Spring Harbor Laboratory Press; 1989. p. 7.3-7.23.
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14. Siebert PD, Larrick JW. PCR MIMICS: competitive DNA fragments for use as internal standards in quantitative PCR. Biotechniques 1993;14:244-9. 15. Nexo E, Jorgensen E, Hansen MR. Human epidermal growth factor: on molecular forms present in urine and blood. Regul Pept 1992;42:75-84. 16. Barnard JA, Graves Deal R, Pittelkow MR, DuBois R, Cook P, Ramsey GW, et al. Auto- and cross-induction within the mammalian epidermal growth factor–related peptide family. J Biol Chem 1994;269:22817-22. 17. Dluz SM, Higashiyama S, Damm D, Abraham JA, Klagsbrun M. Heparin-binding epidermal growth factor-like growth factor expression in cultured fetal human vascular smooth muscle cells. Induction of mRNA levels and secretion of active mitogen. J Biol Chem 1993;268:18330-4. 18. Higashiyama S, Abraham JA, Klagsbrun M. Heparin-binding EGF-like growth factor synthesis by smooth muscle cells. Horm Res 1994;42:9-13. 19. Myers RB, Kudlow JE, Grizzle WE. Expression of transforming growth factor-alpha, epidermal growth factor and the epidermal growth factor receptor in adenocarcinoma of the prostate and benign prostatic hyperplasia. Mod Pathol 1993;6:733-7. 20. Adam RM, Borer JG, Williams J, Eastham JA, Loughlin KR, Freeman MR. Amphiregulin is coordinately expressed with heparin-binding epidermal growth factor-like growth factor in the interstitial smooth muscle of the human prostate. Endocrinology 1999;140:5866-75.
The epidermal growth factor (EGF) system is a rapidly expanding system of growth factors involved in many aspects of normal and cancerous growth. We have developed a method for the quantitation of mRNA coding for all six growth factors activating the human EGF receptor (HER-1) and for the quantitation of mRNA for the receptors HER-1 and its preferred dimerization partner, HER-2. The method is based on the generation of specific RNA standards, which are amplified by reverse transcriptase–polymerase chain reaction (RT-PCR) with the sample RNA and a set of calibrators. The resulting calibration curve is used to quantitate the unknown samples, which require only a single RT-PCR reaction. Our method has the advantage that quantitation is based on coamplification of an internal RNA standard, thereby controlling both the PCR and RT reactions. In addition, the RNA standards for all growth factors and receptors are combined in a single RT reaction, which minimizes variation and allows the quantitation of all eight mRNA species with only 0.1 µg RNA. This makes the method suitable for studies in which the supply of material is limited. The developed method has enabled us to demonstrate that prostate stromal cells in primary culture express EGF, heparin-binding EGF (HB-EGF), amphiregulin, betacellulin, and epiregulin as well as the HER-1 and HER-2 receptors, whereas no transforming growth factor-α mRNA is found. Furthermore, activation of the EGF system in these cells by stimulation with HB-EGF or EGF in mitogenic doses causes a selective increase in the expression of amphiregulin and HB-EGF mRNA (more than 15-fold and 25-fold, respectively), whereas there is no increase in the expression of mRNA for the other growth factors or receptors. In accord with the increase in amphiregulin mRNA, the amount of amphiregulin peptide released from the cells is also increased. The selective induction of amphiregulin and HB-EGF by growth factor stimulation may represent a mechanism to amplify the initial growth factor signal in prostate stromal cells. (J Lab Clin Med 2000;136:209-17) Abbreviations: dATP = deoxyadenosine triphosphate; dCTP = deoxycytidine triphosphate; dGTP = deoxyguanosine triphosphate; dTTP = deoxythymidine triphosphate; EGF = epidermal growth factor; EGTA = ethyleneglycol-bis (β-aminoethylether)-N,N,N´,N´-tetraacetic acid; ELISA = enzyme-linked immunosorbent assay; HB-EGF = heparin-binding epidermal growth factor-like growth factor; HEPES = N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid; HER = human epidermal growth factor receptor; PBS = phosphate-buffered saline solution; PIPES = piperazine-N,N ’- bis (2-ethanesulfonic acid); RT-PCR = reverse transcriptase–polymerase chain reaction; TGF-α = transforming growth factor-α From the Department of Clinical Biochemistry, AKH, Aarhus University Hospital. Supported by the Danish Medical Research Council, the Danish Cancer Society, the Danish Cancer Research Foundation, and the Clinical Research Unit of the Danish Cancer Society, Aarhus, and as part of the Aarhus University Novo Nordic center for research in growth and regeneration. Submitted for publication January 14, 2000; revision submitted April 24, 2000; accepted May 11, 2000.
Reprint requests: Boe Sandahl Sørensen, Department of Clinical Biochemistry, AKH, Aarhus University Hospital, Nørrebrogade 44, DK8000 Aarhus C, Denmark. Copyright © 2000 by Mosby, Inc. 0022-2143/2000 $12.00 + 0 5/1/108753 doi:10.1067/mlc.2000.108753
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Originally the EGF system was considered to consist of one receptor (HER-1) and one ligand, EGF. During the last few years five additional growth factors activating HER-1 have been described, together with additional receptors. Among these receptors, HER-2 is the preferred dimerization partner for HER-1.1 The five additional ligands are betacellulin, HB-EGF, epiregulin, TGF-α, and amphiregulin.2 It is well known that activation of the EGF system is of importance both for growth and for differentiation of cells. Lack of activity in the system is incompatible with life, and increased activity in the system is a common feature of malignant growth.3 Despite this general knowledge on the importance of the system, the relative impact of the many ligands of the EGF system is not known. Investigation of this area is not an easy task. The system is considered to act locally as paracrine and autocrine growth factors; for this reason there is a need for reliable analyses that can determine the expression of the EGF system in minute amounts of tissue or in cultured cells. One of the organs believed to be influenced by the EGF system is the prostate gland.4 Growth of the prostate can lead either to a hyperplastic growth of the gland or to prostate cancer. Growth factors from the EGF system have been shown to be responsible for autocrine stimulation of prostate cancer cells.5 Furthermore, stimulation with EGF induces proliferation of epithelial cells derived from the prostate.6 Proliferation of stromal cells from the prostate has also been shown to be increased by growth factor stimulation.7 In the prostate the EGF system has also been suggested to play an important role for stroma-epithelium interactions.8 To further explore the role of the EGF system, our aim was to develop a method that makes it possible to quantitate the expression of all of the HER-1–activating ligands, of HER-1, and of its preferred heterodimerization partner, HER-2, in prostate stromal cells grown in primary culture. In addition, we wanted to investigate the regulation of the expression of the EGF system during growth factor–induced proliferation. Competitive RT-PCR with an RNA standard that is amplified together with the sample RNA is a sensitive method for mRNA quantitation. Previously we have shown that the method can be refined by inclusion of a set of calibrator RNA samples and development of a calibration curve.9 In the present article we extend this method to include a quantitation of all of the HER-1 activating growth factors and two receptors from the EGF system in human beings. Employing this method, we demonstrate that prostatic stromal cells express all of the ligands examined except TGF-α and all of the receptors investigated (HER-1 and HER-2). We demonstrate a selective induc-
tion of amphiregulin and HB-EGF on stimulation with EGF or HB-EGF. METHODS Cell isolation and culture conditions. Primary cultures of prostate stromal cells were established from human prostate tissue isolated by transurethral resection. Characterization and culturing of the prostate stromal cells was performed as we have previously described,10 according to the method of Zhang et al.11 The cells were cultured in the following serum-containing medium: MCDB 131 (Sigma), 15% heat-inactivated and calcium-stripped horse serum, 1% penicillin/streptomycin, 1% nonessential amino acids, 10 mmol/L HEPES (all from Biological Industries), 10 µg/mL transferrin, 5 µg/mL human insulin (both from Boehringer Mannheim), 5 ng/mL sodium selenite, 0.1 µmol/L estradiol, 0.1 µmol/L dexamethasone (all from Sigma), 1 ng/mL basic fibroblast growth factor, and 0.1 ng/mL EGF (both from Biomol). Cells growing out from tissue pieces were detached by trypsination and passed. In brief, for determination of cell proliferation, 2000 cells/cm2 were seeded in 96-well microtiter trays and grown for 1 day in the serum-containing medium described above. The cells were then starved for 2 days in serum-free medium consisting of MCDB 131 medium (Sigma), 0.1% wt/vol human albumin (Behringwerke), 1% penicillin/streptomycin, 10 mmol/L HEPES, 10 µg/mL transferrin, 2,5 µg/mL human insulin, and 5 ng/mL sodium selenite. Finally, the cells were stimulated with 1 nmol/L HBEGF or 1 nmol/L EGF for 24 hours. Proliferation was measured with the MTT assay (Promega) according to the manufacturer’s instructions. The HCV29 cell line used as calibrator in the mRNA quantitation method was from the Fibiger Institute, Denmark. This cell line is of urothelial origin, characterized by its immortalized growth pattern, and histologically graded as tumor grade two.12 RNA isolation. Cells in third passage were seeded in tissue culture dishes (Greinar) at a density of 2000 cells/cm2 and cultured in the serum-containing medium described above. After 72 hours the medium was changed to the previously described serum-free medium, and the cells were kept in this medium for 48 hours. Cells were stimulated with 1 nmol/L HB-EGF (R&D Systems) or 1 nmol/L EGF (Oncogene) in this medium. The cells were detached from the culture flasks by scraping and were collected by centrifugation at 1500 rpm for 5 minutes. Total RNA was isolated with the Purescript kit (Gentra) by following the instructions provided by the manufacturer. After purification, the concentration and purity of the RNA preparations were analyzed by optical density at wavelengths of 260 and 280 nm (GeneQuant II; Pharmacia Biotech), as previously described.13 Generation of internal RNA standards. RNA internal standards were prepared by modification of previously described methods.9,14 A segment of a 0.6 kb EcoRI-BamHI fragment (spacer DNA) of the v-erb B oncogene (Clontech) was PCR amplified with two composite primers (illustrated in Fig 1). One primer contained at the 5´ end the T7 RNA polymerase promotor sequence followed by one of the genespecific primer sequences and at the 3´ end a region hybridizing to the spacer DNA sequence. The other primer was com-
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posed of a stretch of 20 T nucleotides at the 5´ end followed by the other gene-specific primer and at the 3´ end a sequence that hybridized to the spacer DNA. A PCR was performed that contained 1 ng of the spacer DNA, 10 pmol of each of the two composite primers, 1.5 mmol/L MgCl2, 0.2 mmol/L deoxyribonucleoside triphophates, and 2.5 U Taq DNA polymerase (Pharmacia). A Perkin Elmer thermal cycler was used with an amplification profile of 1 minute at 94°C, 30 seconds at 65°C, and 90 seconds at 72°C (an initial heating to 94°C for 3 minutes was performed). Thirty cycles were conducted, and at the end the samples were extended for 7 minutes at 72°C. A single DNA band of the correct size was verified by gel electrophoresis (data not shown). RNA internal standard was obtained by incubation of 1 µg of the PCR product with 40 U of T7 RNA polymerase (Boehringer Mannheim) in the buffer supplied by the manufacturer for 2 hours at 37°C. DNA was digested with ribonuclease-free DNase I (Promega) followed by extraction with water-saturated phenol/chloroform (24:1). RNA in the water phase was precipitated by the addition of 1/10 vol of 4 mol/L NaCl and 2.5 vol of 96% ethanol. The RNA was recovered by centrifugation for 15 minutes at 14,000 rpm in an Eppendorf centrifuge and washed with 1 mL 70% ethanol. The RNA pellet was redissolved in nuclease-free water and passed through a spin column (Chromaspin, Clontech) to remove free nucleotides, and finally the RNA was quantitated by spectrophotometry.13 PCR products were not obtained in the absence of reverse transcription, demonstrating the absence of contaminating DNA in the RNA standards (data not shown). By designing the composite primers to hybridize to different positions on the spacer DNA, it was possible to produce RNA internal standards with suitable sizes. The sizes of the internal RNA standards were adjusted according to the sizes of the mRNA targets, resulting in size differences between 50 and 200 bp for the eight mRNA species investigated. Internal RNA standards were generated for EGF, TGF-α, amphiregulin, HB-EGF, betacellulin, epiregulin, HER-1, and HER-2, and the sizes of the RT-PCR products originating from the internal RNA standards and the corresponding mRNAs are presented in Table I together with the sequences of the gene-specific primers used. Quantitative RT-PCR. Calibrators for the quantitative RTPCR assays were obtained from a pool of total RNA isolated from the urothelial cell line HCV29. This RNA was diluted in water to give calibrators with the following RNA concentrations: 1 µg/µL, 750 ng/µL, 500 ng/µL, 250 ng/µL, 100 ng/µL, 75 ng/µL, 50 ng/µL, 25 ng/µL, 10 ng/µL, 8 ng/µL. The HCV29 cell line was chosen as calibrator because it expresses all of the mRNA species investigated at a sufficiently high concentration for quantitation. The absolute concentrations of the specific mRNAs in the HCV29 calibrator RNA were determined by competitive RT-PCR. In brief, a fixed amount of RNA standard was coamplified with a dilution series of the calibrator RNA (RT-PCR conditions were as described below). The intensities of the bands originating from the RNA standard and the specific mRNA in the calibrator were plotted against the amounts of calibrator used. By this method an amount of calibrator was identified in which equal amounts
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Fig 1. Schematic illustration of the generation of RNA standards. One primer consists of the T7 RNA polymerase promotor sequence (T7), a gene-specific primer sequence (GS1), and a region hybridizing to the spacer DNA (HYB). The other primer is composed of a region of 20 T nucleotides (T20), the other gene-specific primer sequence (GS2), and a region hybridizing to the spacer DNA (HYB). A DNA molecule is generated by PCR, and in vitro transcription of this DNA gives rise to the RNA standard, which has incorporated the gene-specific primer sequence (GS1), the complementary sequence of the other gene-specific primer (GS2), and a tail of 20 A nucleotides (A20). GS1 and GS2 represent the gene-specific primers, and Table I gives the sequences of all eight pairs of gene-specific primers used.
of RT-PCR products from the specific mRNA and the RNA standard were generated. In this amount of calibrator the content of the specific mRNA equals the amount of RNA standard added. Because the RNA standard was present in a known amount, this made it possible to find the absolute mRNA concentrations in the calibrator. Gel electrophoresis, scanning of bands, and correction for differences in the sizes of target and standard bands were as described below. In the quantitative assays described in the present article, a fixed amount of each of the eight internal RNA standards was combined with the sample RNA to be analyzed and to a set of the HCV29 RNA calibrators, followed by RT-PCR amplification. RNA (0.1 µg) was mixed with the eight different RNA standards in a mixture of 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 6.25 mmol/L MgCl2, 1 U/µL RNase inhibitor, 1 mmol/L deoxyribonucleoside triphosphates (dATP, dCTP, dTTP, dGTP), 2.5 µmol/L of a 16 mer d(T)16 oligonucleotide primer, and 2.5 U/µL reverse transcriptase in a reaction mixture of 20 µL (all reagents from Perkin-Elmer). Reverse transcription was performed by incubation at 42°C for 30 minutes. A 2.5 µL sample of the reaction mixture was used for PCR in a total volume of 25 µL containing 50 mmol/L KCl, 10 mmol/L Tris-HCl (pH 9.0), 1.5 mmol/L MgCl2, 0.2 mmol/L deoxyribonucleoside triphosphates (dATP, dCTP, dTTP, dGTP), 1.25 U of Taq polymerase (Pharmacia), and the amount of primer shown in Table I. PCR was performed in a Perkin Elmer thermal cycler with the following standard cycling parameters: 94°C for 1 minute, annealing temperature (presented in Table I) for 30 seconds, and 72°C for 90 seconds (initially the samples were dena-
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Table I. Primer sequences and sizes of RT-PCR products Gene-specific primers
Target size
Standard size
Amphiregulin
5´-TTGATACTCGGCTCAGGCC-3´ 5´-CTACTGTCAATCATGCTGTGA-3´ (4 pmol/60°C/39 cycles)
539 bp
345 bp
HB-EGF
5´-GATGGTTGTGTGGTCATAGGT-3´ 5´-TGGCTGCAGTTCTCTCGGC-3´ (10 pmol/62°C/35 cycles)
449 bp
340 bp
Betacellulin
5´-CTGCAAAGTGCCTTGCTCA-3´ 5´-TGACTAGTAATCCTGGTGAC-3´ (10 pmol/59°C/33 cycles)
251 bp
315 bp
Epiregulin
5´-AATGTAACTCCACTGTTCTCC-3´ 5´-CCTTCTCCCATATGAACAAGA-3´ (10 pmol/59°C/33 cycles)
357 bp
418 bp
EGF
5´-AGCAATTGGTGGTGGATG-3´ 5´-ACTCTTTGCAAAAGTTGTC-3´ (5 pmol/54°C/35 cycles)
103 bp
163 bp
TGF-α
5´-GCCCGCCCGTAAAATGGTCCCCTC-3´ 5´-GTCCACCTGGCCAAACTCCTCCTCTGGG-3´ (2.5 pmol/70°C/32 cycles)
528 bp
357 bp
HER-1
5´-GAGAGGAGAACTGCCAGAA-3´ 5´-GTAGCATTTATGGAGAGTG-3´ (25 pmol/57°C/30 cycles)
454 bp
314 bp
HER-2
5´-AGATGTTCGGCCCCAGCCCCCTT-3´ 5´-GTGGAGCCCCCCGCTCTGGTG-3´ (10 pmol/68°C/30 cycles)
272 bp
420 bp
Items in parentheses contain the following information: primer concentration/annealing temperature/PCR cycles.
tured at 94°C for 3 minutes). After the number of cycles used for each of the PCR products (Table I), the PCR products were extended for 7 minutes at 72°C. PCR products were analyzed by electrophoresis in a 2% agarose gel (Meda). The intensities of the bands were determined by computer scanning with the Gel Doc 1000 system (BioRad) and the molecular analyst software (BioRad). Intensities of the target and standard bands were corrected for differences in size of the RT-PCR products, because binding of ethidium bromide is dependent on DNA size. The ratio of the amount of PCR product originating from the target mRNA and the internal standard RNA was determined for each of the calibrators. This ratio was plotted against the amount of the specific mRNA present in each calibrator, thereby generating a calibration curve. By use of the calibration curve, the ratio of the PCR products from target and internal standard RNA obtained from the unknown samples could be used to quantitate the specific mRNAs. Samples exceeding the standard curve were diluted in water before repeated analysis. Coefficients of variation of the calibrated quantitative assays ranged between 9% and 21%, as is typical for this type of assay. Equal efficiency of the RT-PCR amplification of target mRNA and corresponding internal RNA standard was demonstrated by RT-PCR amplification of mixtures of the two molecules with decreasing numbers of cycles (data not shown). The identity of all PCR products was verified by sequencing (ABI Prism 310; Perkin Elmer).
Peptide expression. Cells in fourth or fifth passage were seeded in tissue culture flasks (T175; NUNC) at a density of 2000 cells per cm2 and cultured in the serum-containing medium described above. After 48 hours the medium was changed to serum-free medium. Forty-eight hours later, 1 nmol/L HB-EGF or 1 nmol/L EGF was added to the medium and the cells were cultured for an additional 96 hours. The medium was removed and filtered through a 0.22µm filter (Gelman Sciences), concentrated 10-fold by lyophilization, and dialyzed (cut off value of 3.5 kd) for 48 hours in PBS containing 0.1% bovine albumin (Sigma). Cells were washed in ice-cold PBS and scraped off. The cells were isolated by centrifugation at 1500 rpm for 8 minutes at 4°C before being stored at –20°C. After thawing, 0.3 mL of homogenization buffer (pH 7.4) containing 10 mmol/L PIPES (Sigma), 1 mmol/L EGTA (Sigma), 3 mmol/L MgCl2, 400 mmol/L NaCl, and complete protease inhibitor (Boehringer Mannheim) was added to the cells, and the suspension was sonicated three times for 10 seconds before centrifugation at 20,000g for 40 minutes at 4°C. The supernatant was isolated (supernatant I), and the pellet was suspended in 0.3 mL homogenization buffer containing 2% Triton X-100 and sonicated. The suspension was left overnight at 4°C and centrifugated as indicated above. The supernatant was isolated (supernatant II) and stored at –20°C before analysis. The concentration of growth factors in the cell supernatant
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A Fig 2. RT-PCR coamplification of mRNA and internal RNA standards from the EGF system. Calibrator RNA (RNA from the HCV29 cell line) was RT-PCR amplified with internal RNA standard. Lanes 1 through 8 represent HER-1, HER-2, amphiregulin, betacellulin, EGF, epiregulin, HB-EGF, and TGF-α, respectively (DNA sizes are presented in Table I). M is the size marker (φX174 DNA digested with Hae III).
and conditioned medium was determined by traditional sandwich ELISA technique. Amphiregulin was measured with the MAB-262 (capture antibody) and the AB-262-NA (detecting antibody), both from R&D Systems. Recombinant human amphiregulin 262-AR-100 (R&D Systems) was used as calibrator. EGF was measured with the rabbit anti-mouse IgG1 (Z0013-DAKO), the anti human EGF Ab-1 (GF01, Calbiochem) as capture antibody, and a polyclonal rabbit antihuman EGF antibody as detecting antibody.15 Recombinant human EGF (Oncogene) was used as calibrator. Protein concentrations of the supernatants were determined by the BCA protein assay (Pierce).
B
RESULTS Quantitation of mRNA expression from the EGF system.
Quantitative RT-PCR based methods were developed for EGF, amphiregulin, TGF-α, HB-EGF, betacellulin, epiregulin, HER-1, and HER-2. Primer sequences, annealing temperatures, numbers of PCR cycles, and sizes of the RT-PCR products obtained from the genespecific mRNAs and internal RNA standards for the members of the EGF system are presented in Table I. Fig 2 shows all eight target and corresponding internal RNA standard pairs investigated. Fig 3, A, shows the RT-PCR amplification of HBEGF mRNA and the corresponding internal RNA standard as a representative example of the quantitative mRNA assay. In brief, in the quantitative assay, a mixture of all eight internal RNA standards was added to both the RNA samples (0.1 µg) and the calibrators, and a single reverse transcription reaction was performed. This cDNA was used as the template for all eight specific PCR reactions. A calibration curve was generated by plotting the ratio of the intensities of the RT-PCR bands from HB-EGF mRNA and internal RNA standard against the amount of the specific mRNA in the individual calibrators. Fig 3, B, and Fig 3, C, show the gel image and calibration curve, respectively, of the quantitative HB-EGF mRNA assay.
C Fig 3. Method for quantitation of mRNA from the EGF system. A, Schematic illustration of the PCR amplification of HB-EGF mRNA and standard RNA with the same primer pair (P1 and P2) shown as an example. B, Gel showing the calibration curve obtained by RTPCR amplification of serial dilutions of calibrator RNA combined with a fixed amount of internal RNA standard. Lanes 1 through 10 represent 1.0 µg, 0.75 µg, 0.5 µg, 0.25 µg, 0.1 µg, 0.075 µg, 0.05 µg, 0.025 µg, 0.01 µg, and 0.008 µg, respectively. Lane 11 is a control with standard RNA but without calibrator RNA. C, Calibration curve based on scanning of the gel in B. The ratios of the RT-PCR products from HB-EGF mRNA and internal RNA standard are depicted as a function of the amount of HB-EGF mRNA present in the added calibrator RNA samples. The standard curve is obtained by linear regression (y = 1.0x + 19.0).
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A
B Fig 4. Selective induction of HB-EGF and amphiregulin expression by growth factor stimulation of prostate stromal cells. A, Prostate stromal cells were kept in minimal essential medium for 48 hours, and the mRNA expression of growth factors and receptors from the EGF system was determined. Each bar represents the mean expression level of four individual experiments (standard deviation is indicated). B, The cells were incubated in the presence or absence of 1 nmol/L HB-EGF for 48 hours, and the expression of growth factors and receptors was determined. The induction of mRNA expression is presented as the ratio between the mRNA expression in the HB-EGF stimulated and unstimulated cultures (ratio of the mean expression in two stimulated and two unstimulated cultures). The experiments presented in A and B are representative examples of three independent experiments. The inserted gel picture shows representative RT-PCR amplification data illustrating the coamplification of a fixed amount of HB-EGF internal RNA standard and HB-EGF mRNA from RNA isolated from HB-EGF treated and control cells (lanes 1 and , respectively). For the actual quantitation, calibrators would be included and a standard curve would be generated (as presented in Fig 3).
HB-EGF and amphiregulin mRNA is induced by stimulation with HB-EGF or EGF. Prostate stromal cells grown
in primary cultures and kept in a serum-depleted medium for 48 hours express mRNA for all the growth factors and receptors examined except TGF-α. The expression was lowest for amphiregulin, while comparable expression was observed for betacellulin, epiregulin, EGF. and HB-EGF (Fig 4, A). The level of mRNA expression for the two receptors was slightly higher than the expression for the growth factors (Fig 4, A).
Prostate stromal cells were grown in the absence or presence of HB-EGF or EGF (both 1 nmol/L) for 48 hours. In Fig 4, B, the levels of induction of growth factor and receptor mRNA expression after stimulation with HB-EGF are presented as mRNA expression in the stimulated cells divided by expression in the unstimulated cells. This demonstrates that a selective induction of amphiregulin and HB-EGF mRNA takes place. A similar result was observed after 24 hours of incubation. None of the other ligands and none of the
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Fig 5. Induction of amphiregulin peptide by HB-EGF stimulation of prostate stromal cells. Prostate stromal cells were kept in minimal essential medium for 96 hours in the presence or absence of 1 nmol/L HB-EGF. Cells and medium from the cells were isolated, and the amount of amphiregulin and EGF peptide was determined as described in Methods. During the experiment there was a 40 % increase in cell number of the HB-EGF–treated cells. The peptide concentrations of the HB-EGF–treated cells were adjusted to account for this increase in cell number. No EGF was found in the medium of either control or HB-EGF–stimulated cells. Shown is a representative example. The experiment was repeated twice with similar results.
Fig 6. EGF- and HB-EGF–stimulated proliferation of prostate stromal cells. Cells were serum-starved for 48 hours and then grown for 96 hours with either 1 nmol/L HB-EGF or 1 nmol/L EGF or in the absence of growth factor (control cells). Proliferation was determined by the MTT assay. Data are presented as fold increase in proliferation as compared with the proliferation of the control cells. Each column represents the mean value of 16 colonies (standard deviation indicated). There was a significant increase in proliferation after EGF and HB-EGF treatment (P < .01) as compared with the control cells. The experiment was repeated three times, and a representative example is shown.
receptors examined increased after stimulation with HB-EGF (Fig 4, B). Similar results were obtained by stimulation with EGF (data not shown).
stimulated the growth of prostate stromal cells in primary cultures (P < .01). Fig 6 shows the results obtained after growth factor stimulation for 96 hours. The figure presents the mean ± SD values for 16 individual cultures. Similar results were obtained in two additional experiments that used different primary cultures.
Induction of amphiregulin peptide by HB-EGF stimulation. The concentrations of amphiregulin and EGF pro-
tein were determined in both culture medium and cells after 96 hours in serum-free medium with or without 1 nmol/L HB-EGF. The control cells contained comparable amounts of EGF and amphiregulin (Fig 5). No EGF was detected in the medium, whereas a total of 140 fmol amphiregulin was measured in the 40 mL of medium collected from the control cells. The amount of EGF did not increase on stimulation with HB-EGF. Likewise, the concentration of amphiregulin in the cells was not affected by HB-EGF. In contrast, the concentration and the total amount of amphiregulin in the medium increased to 240% after HB-EGF treatment (340 fmol in the 40 mL medium). Correcting for the increase in cell number (40%), we found the amount of amphiregulin released per cell to increase to 170% of the control level after treatment of the cells with HB-EGF (Fig 5). All protein and mRNA quantitations presented were performed on primary cultures from the same donor. Similar results were found in experiments performed on primary cultures obtained from two different donors. HB-EGF and EGF stimulate proliferation of prostate stromal cells. The mitogenic potential of the HB-EGF and
EGF doses used to induce mRNA expression of HBEGF and amphiregulin was investigated. Equimolar amounts (1 nmol/L) of HB-EGF or EGF significantly
DISCUSSION
Because of the complexity of the EGF system, refined methods are required so that the mRNA expression of several members of the family can be investigated simultaneously. We report a new method for quantitating the mRNA expression of the six HER-1 activating growth factors as well as HER-1 and its preferred heterodimerization partner, HER-2. This calibrated RT-PCR reaction has the advantage that only a single PCR reaction is required to analyze each mRNA. Imprecision is minimized by our procedure, in which all eight RNA standards are combined with the sample RNA before reverse transcription, thereby generating a common cDNA sample that can be used for all eight specific PCR reactions. A further advantage is that only a 0.1-µg sample of RNA is required for quantitation of all eight mRNA species. This makes the method suitable for clinical applications in which limited material is available. The design of the assay makes it possible to include control samples, which can be used to monitor the performance of the assay over time. An important prerequisite for correct quantitation by the assays
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described here is that the RNA standard and the target mRNA are RT-PCR anplified with the same efficiency. This can be verified by demonstrating that the ratio between the target and standard RT-PCR products from RNA samples remains unaffected when the cycle number in the PCR amplification is decreased.9 Growth of the prostate as observed in both benign prostatic hyperplasia and prostate cancer represents important clinical problems. However, only limited information is available regarding the expression of the components of the EGF system in human prostate stromal cells and the regulation of their expression during growth factor–induced proliferation. Our new method has enabled us to demonstrate for the first time that five of the six EGF receptor ligands, as well as the two receptors examined, are expressed in primary cultures of prostate stromal cells. We further demonstrate that stimulation of the cells with mitogenic concentrations of either HB-EGF or EGF selectively increases the expression of HB-EGF and amphiregulin by more than 25-fold and 15-fold, respectively. Growth factor stimulation had no effect on mRNA expression of the HER1 and HER-2 receptors, which indicates that induction of the EGF system in prostate stromal cells is mediated at the growth factor level rather than by induction of the HER-1 and HER-2 receptors. In accord with the amphiregulin mRNA induction on stimulation with growth factor, the amphiregulin protein level was also increased in the medium from the cell cultures, as determined by ELISA. A suitable assay for quantitation of the other induced growth factor (HBEGF) at the protein level was not available. Both HBEGF and amphiregulin are distinguished from the other ligands from the EGF system by their heparin-binding ability, suggesting that this subgroup of growth factors might play a special role in the induction of growth factors after growth factor stimulation of prostate stromal cells. At present the biologic significance of this induction is not known, but we have demonstrated that exogenous HB-EGF (the present study) and amphiregulin10 induce proliferation of prostate stromal cells. Autoinduction of growth factors from the EGF system has been shown to occur in a few other cell types such as keratinocytes, intestinal cells, and fetal vascular smooth muscle cells.16-18 In one study, four of the six ligands were investigated, and it was reported that in growth factor–treated keratinocytes, all of the growth factors examined were induced.16 HB-EGF and amphiregulin were induced to the highest level, whereas betacellulin and TGF-α were induced to a lower degree. In contrast, we found that in primary cultures of human prostate stromal cells, the mRNA induction is restricted to HB-EGF and amphiregulin. These two studies demonstrate that in different cell types, differ-
ences exist with respect to the induction of ligands from the EGF system. A consequence of the growth factor induction reported here might be that the signal from the initial growth factor stimulation is amplified and sustained, thereby promoting autocrine growth of the stromal cells. In addition, our observation that amphiregulin is secreted to the culture medium of the prostate stromal cells suggests that the increased growth factor expression is also responsible for mediating paracrine stimulation of the prostatic epithelium. This possibility is in accord with previous studies showing a high expression of HER1 in the basal epithelial cell layer of the prostate,19 which is in close approximation to the stromal cells. This concept is further supported by recent findings showing that amphiregulin and HB-EGF are coordinately regulated in prostate smooth muscle cells and are likely to be involved in signaling between the smooth muscle cells and the epithelial cells of the prostate.20 In conclusion, we present a method whereby the mRNA expression from all six HER-1 ligands, as well as the HER-1 and HER-2 receptors, can be quantitated simultaneously in a limited amount of RNA (0.1 µg). The method can easily be applied to other studies in which the simultaneous quantitation of several mRNA species is important. The developed method has enabled us to demonstrate that growth factor activation of prostate stromal cells grown in primary culture results in a selective induction of amphiregulin and HB-EGF mRNA. We thank Klaus Møller-Ernst Jensen for providing the human prostate tissue. We also thank Birgit Westh Mortensen, Alice Villemoes, and Inger Marie Jensen for technical assistance.
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